Identification and characterization of a population of motile neurons in long‐term cortical culture

Abstract

The specific phenotypes and progression to maturity of primary cortical neurons in long-term culture correlate well with neurons in vivo. Utilizing a model of neuronal injury in long-term cultures at 21 days in vitro (DIV), we have identified a distinct population of neurons that translocate into the injury site. 5-bromo-2'-deoxyUridine (BrdU) incorporation studies demonstrated that neurons with the capacity to translocate were 21 days old. However, this motile ability is not consistent with the traditional view of the maturation and structural stability of neurons in long-term culture. Therefore, we examined the neurons' cytoskeletal profile using immunocytochemistry, to establish relative stage of maturation and phenotype. Expression of marker proteins including beta-III-tubulin, alpha-internexin, NF-L and NF-M, tau and L1 indicated the neurons were differentiated, and in some cases polarized. The neurons did not immunolabel with NF-H or MAP2, which might suggest they had not reached the level of maturity of other neurons in culture. They did not express the microtubule-associated migration marker doublecortin (DCX). Cytoskeletal disrupting agents were used to further investigate the role of the microtubule cytoskeleton in translocation, and microtubule destabilization significantly enhanced aspects of their motility. Finally, molecular guidance cues affected their motility in a similar manner to that reported for both axon guidance and early neuron migration. Therefore, this study has identified and characterized a population of motile neurons in vitro that have the capacity to migrate into a site of injury. These studies provide new information on the structurally dynamic features of subsets of neurons.

Cyclosporin‐A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury

“…Neuron cultures were prepared as reported previously (Dickson et al, 2000). This culture technique produces nerve cells that are relatively mature and reflective of the adult CNS (Chuckowree and Vickers, 2003;Chung and West, 2004;King et al, 2006;Haas et al, 2007). Briefly, neocortical tissue was removed from embryonic day (E)18 (sperm-positive day ¼ E1) Hooded Wistar rat embryos and incubated in sterile 10 mM HEPES buffer (378C).…”

Influence of microchannel geometry on device performance and electrophysiological recording fidelity during long-term studies of connected neural populations

“…While it has been shown that small populations of cultured neurons can migrate in response to specific stimuli, the large majority of cultured neurons are considered non-motile. 35,36 Conversely, astrocytes have been shown to migrate readily even in the absence of stimulation, 37 and therefore we would expect a higher ratio of non-confined astrocytes to neurons if post-seeding migration was dominant. This suggests that cell confinement may not be a critical factor if each chamber is seeded with cells from the same initial population, however in instances where two distinct cell populations are seeded in opposing chambers, reducing microchannel height to prevent cellular cross-contamination is critical.…”

Genotype-Specific Differences between Mouse CNS Stem Cell Lines Expressing Frontotemporal Dementia Mutant or Wild Type Human Tau

“…We saw high levels of transgenic tau protein throughout neuritic projections in differentiated neurospheres from both human tau transgene genotypes. The presence of tau in the filopodia-spines of developing cells [74], [83] suggests that the phenomenon correlates with a dynamic morphology and does not necessarily indicate pathological injury.…”